Studies of the structure–function relationships of Neurospora crassa pyruvate kinase: interaction with blue dextran – Sepharose and Cibacron blue 3G-A

Abstract

Blue dextran--Sepharose and Cibacron blue 3G-A interact with pyruvate kinase of Neurospora crassa. The enzyme is readily released from the substituted Sepharose column by elution with 0.17 M potassium phosphate buffer (pH 7.9), or 2 mM fructose 1,6-diphosphate (FDP), but not with either of the substrates, ADP and phosphoenolpyruvate (PEP), at 2 mM. Cibacron blue 3G A is a noncompetitive inhibitor of pyruvate kinase with respect to both substrates. It appears to compete with the allosteric effector, FDP, for binding to the enzyme surface. A lack of elution of the enzyme from the immobilized blue dextran matrix by adenine nucleotides and the absence of a difference spectrum in the 650- to 700-nm range suggest that a "dinucleotide-fold" substructure is not implicated in the dye binding sites on pyruvate kiase. The interaction of Cibacron blue 3G-A and this enzyme can be followed fluorometrically; incremental additon of the dye to the enzyme solution results in a progressive decrease in the fluorescence of surface tryptophanyl residues. The quenching of fluorescence of exposed aromatic groups is subject to reversal following addition of FDP to the pyruvte kinase--Cibacron blue complex.

Interaction of 3-hydroxybenzoate-6-hydroxylase with cibacron blue

“…An alternative explanation is the induction of a local conformational change in the enzyme that leads to the exposure of tryptophan residues to solvent thereby reducing the quantum yield of emission fluorescence. Quenching of protein fluorescence by cibacron blue has been also reported for various proteins and enzymes such as p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens and pyruvate kinase from Neurospora crassa [10,22,27]. The concentration-dependent quenching of the intrinsic fluorescence of the enzyme by cibacron blue provided a convenient method for the quantification of binding parameters under various conditions.…”

Some aspects of the regulation of pyruvate kinase levels in Neurospora crassa

“…Immunoadsorption, immunoprecipitation, and SDS -polyacrylamide gel electrophoresis Pyruvate kinase was isolated and polyclonal antibodies raised against the purified preparation, as described previously (Kapoor and O'Brien 1980;Kapoor and Bishop 1982). For some experiments the anti-PK IgG fraction was purified further by passage through a column containing PK coupled to Affigel-10.…”